This invention relates to liquid two-phase extraction systems, particularly, to systems for the affinity partitioning of proteins. This invention also relates to a method of separating proteins using a two-phase protein extraction system.
Aqueous two-phase affinity partitioning methods have been described for the separation of macromolecules such as proteins and nucleic acids, cell particles, and intact cells on a large scale. These types of systems are described, e.g., in Albertsson, Partition of Cell Particles and Macromolecules, John Wiley and Sons, New York, 1986; and Walters et al., Partitioning in Aqueous Two Phase Systems, Academic Press, Inc., 1985.
Partition of enzymes and other proteins between two liquid aqueous phases can be strongly influenced by specific or group specific ligands bound to a water-soluble polymer. A protein ligand covalently attached to a phase-forming polymer can cause that ligand to predominately partition into one of the phases. Affinity partitioning occurs when the partition characteristics of protein are altered due to the interaction of the protein with its asymmetrically partitioned ligand.
Several low cost two-phase systems are known which can handle protein separations on a large scale. These systems use polyethylene glycol (PEG) as the upper phase-forming polymer and crude dextran (as disclosed in Kroner et al., Biotechnology Bioengineering, 24, 1015-1045, 1982), a concentrated salt solution (as disclosed in Kula et al., Adv. Biochem. Bioeng. 24, 73-118, 1982) or hydroxypropyl starch as disclosed in Tjerneld et al., Biotechnology Bioengineering 30, 809-816, 1987, as the lower phase-forming polymer. Two of these systems, the PEG/salt and PEG-crude dextran are seldom used for affinity partitioning. Affinity partitioning cannot take place efficiently in a PEG/salt system due to the effects of the high salt concentrations, whereas the high viscosity of the PEG/crude dextran and/or high cost of fractionated dextran system makes large scale separations difficult.
One of the most successful systems for affinity partitioning thus far on a large scale is the PEG/hydroxypropyl starch system using textile dyes that are covalently bonded to PEG as affinity ligands. Affinity partitioning in the PEG/dextran system is carried out by coupling monochloro-triazine textile dyes to the upper phase polymer (PEG). It utilizes a protein-ligand (i.e., substrate analog, antibody, or textile dye) that is covalently coupled to one of the phase-forming polymers to ensure that the ligand is partitioned predominately into one phase. Although triazine dyes covalently coupled to PEG can be produced on a large scale, the process requires a chromatographic step and several organic solvent extractions in order to produce an effective affinity ligand. To optimize a particular affinity partitioning system, the dye type and other PEG bound ligands need to be empirically tested.
The patent literature also discusses various methods of two-phase aqueous partitioning.
U.S. Pat. No. 4,684,723 discloses a method for separating and recovering proteins from an aqueous system containing one or more therapeutically active proteins or nucleic acids and a polymer component, preferably PEG, having the ability to create two liquid phases within the liquid system. In this method, a water-soluble inorganic salt is added to the aqueous system thereby separating the system into two or more liquid phases. Such separated phases may be selectively enriched in components of the original aqueous system having differing solubilities in the two resultant liquid phases.
U.S. Pat. No. 4,579,661 discloses purification of a biologically active substance with the aid of a system having at least two immiscible aqueous phases. In the process, insoluble particles, e.g., PEG particles, having an affinity for the biologically active substance and a high coefficient of distribution for one of the aqueous phases are used as ligands to bind the substance. Subsequently, the biologically active substance is liberated from the particles after separating the particle-containing phase from one or more other phases.
U.S. Pat. No. 4,645,929 discloses a method of separating polypeptide fractions of a mixed solution by addition of a soluble charged polymer and a soluble neutral polymer. The charged polymer interacts with one fraction of polypeptides to form a precipitate. Neutral polymer enhances the effectiveness of the charged polymer in precipitating this fraction. By proper selection of the polymer combination, a desired polypeptide fraction can be precipitated from the solution while the other polypeptide fraction remains in solution or vice versa.
U.S. Pat. No. 4,716,219 discloses adsorbents for affinity chromatography of proteins consisting of an electroneutral carrier matrix, a spacer and a triazine coloring substance bonded to the amino group of the spacer.
In contradistinction to the above-described systems, it was desired to find a cost-effective system wherein the dyes could be easily bound to the polymeric phase, without having to carry out the chromatographic and solvent extractions necessary to form the covalent bond in the PEG/hydroxypropyl starch system of the prior art.